The epidermal growth factor receptor (EGFR) and gp185erbB-2 receptor are closely related tyrosine kinases. Despite extensive sequence and structural homology, they display quantitative and qualitative differences in their ability to couple with mitogenic signalling pathways. Utilizing a chimeric EGFR/erbB-2 molecule, a direct comparison of the biological and biochemical events triggered by activation of the erbB-2 and EGFR kinases in NIH/3T3 cells was possible. Under conditions of comparable expression levels in NIH/3T3 cells, the EGFR/erbB-2 chimera conferred increased responsiveness to EGF compared with EGFR, perhaps due to the different intrinsic ability of the two kinases to phosphorylate intracellular substrates. Biochemical analysis of known mitogenic transduction pathways, however, failed to reveal any major difference in the ability of EGFR of gp185erbB-2 to induce tyrosine phosphorylation of PLC-gamma and GAP. Other second messengers, such as PI-3 kinase and c-raf, implicated as substrates for receptor tyrosine kinases, do not efficiently couple with either erbB-2 or EGFR kinase. Results suggest that other, unknown, signal transduction pathways responsible for the different biological effects of EGFR and gp185erbB-2 might exist. To identify and characterize new intracellular substrates, we purified EGF-induced phosphotyrosine proteins from NIH/3T3 fibroblasts overexpressing EGFR. The purified proteins were then used to immunize animals for the production of polyclonal sera. With this antisera we were able to identify six molecular species which phosphorylated all tyrosine residues upon EGF stimulation. V8 protease digestions showed that none were degradation products of the EGFR. Furthermore, the newly identified species do not correspond to any of the known substrates for tyrosine kinase receptors. The polyclonal antisera were also used for screening bacterial expression libraries with which we characterized a number of cDNAs.